Agrobacterium sp. 1502 IPR-08 and its applications

Abstract

The present application relates to the field of microbiology and plant growth promoting bacteria, in particular to a strain of Agrobacterium sp. 1502IPR-08 and its application. The Agrobacterium sp. 1502IPR-08 provided by the present application has a preservation number of CGMCC No. 25883, is gram-negative bacteria, has good nitrogen fixation ability, can convert nitrogen in the air into nitrogen that can be used by plants, increase soil nutrients, promote plant growth, and can successfully colonize in the rhizosphere soil of plants, increase the nitrogen content of the rhizosphere soil, improve the glutamine enzyme activity of the plant root system, and has great application potential.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and plant growth-promoting bacteria, specifically to an Agrobacterium sp. 1502IPR-08 strain and its applications. Background Technology

[0002] Peanuts are one of my country's important oil crops and also one of the country's major export commodities, playing a vital role in promoting the national economy.

[0003] Nitrogen, one of the three essential nutrients for crop growth and development, is a major component of proteins and participates in various physiological processes involved in plant growth and development. Crops have a high nitrogen requirement during growth and development, and a significant amount of nitrogen is removed from the soil during harvest. Therefore, sufficient nitrogen application is necessary in agricultural production. However, farmers often overuse nitrogen fertilizers in pursuit of higher yields, leading to a series of negative problems such as soil compaction, eutrophication of water bodies, groundwater pollution, global warming, photochemical smog, and nitrate accumulation in drinking water and food. These issues seriously hinder sustainable agricultural development and human health. Therefore, reducing the application of chemical nitrogen fertilizers while ensuring crop production needs is crucial for achieving green agricultural development.

[0004] The reduction of molecular nitrogen in the atmosphere to ammonia is a process called nitrogen fixation. Without nitrogen fixation, atmospheric molecular nitrogen cannot be absorbed and utilized by plants. There are three pathways of nitrogen fixation on Earth: biological nitrogen fixation, industrial nitrogen fixation (using high temperature, high pressure, and chemical catalysis to convert nitrogen into ammonia), and high-energy nitrogen fixation (such as the high energy generated by lightning and other high-altitude instantaneous discharges, which can cause nitrogen in the air to combine with hydrogen in water to form ammonia and nitric acid, which are then carried to the ground by rainwater). Biological nitrogen fixation accounts for about 90% of the total nitrogen fixed on Earth each year, demonstrating its crucial role in the Earth's nitrogen cycle. Inoculating plant seeds or roots with nitrogen-fixing bacteria can significantly promote plant growth and increase yield. Simultaneously, it can improve plant nutrition and increase biomass by enhancing soil fertility and antagonizing pathogens. When nitrogen is deficient, plants will increase the contact area between their roots and the soil through root activity; attracting nitrogen-fixing bacteria to colonize the roots and obtain usable nitrogen. Based on their characteristics and their relationship with their hosts, nitrogen-fixing microorganisms can be classified into symbiotic nitrogen fixation, free-living nitrogen fixation, and associative nitrogen fixation. Nitrogen-fixing bacteria and plants exchange nutrients through symbiotic and non-symbiotic mechanisms. Increasing research indicates that the survival characteristics of free-living nitrogen-fixing bacteria broaden their applications; they do not need to colonize the plant interior, providing nitrogen to crops simply by existing in the rhizosphere soil. In the rhizosphere soil, the nitrogen fixation efficiency of free-living nitrogen-fixing bacteria is far higher than that in non-rhizosphere soil. The beneficial interactions between free-living nitrogen-fixing bacteria, soil, and plants can improve plant nitrogen nutrition and increase crop yield. Therefore, utilizing microorganisms for nitrogen fixation is of great significance in reducing nitrogen fertilizer application, increasing crop yields, reducing environmental pollution, and promoting sustainable agricultural development. Summary of the Invention

[0005] The purpose of this invention is to provide a soil bacterium with high nitrogen fixation capacity, which improves plant nitrogen nutrition and increases the nitrogen content of plant rhizosphere soil, and its application.

[0006] This invention provides an Agrobacterium sp. 1502IPR-08, the preservation number of which is CGMCC No. 25883.

[0007] The present invention provides a fermentation product, wherein the fermentation bacteria of the fermentation product include Agrobacterium sp. 1502IPR-08.

[0008] The present invention provides a microbial agent comprising Agrobacterium sp. 1502IPR-08 or the fermentation product.

[0009] Preferably, the effective viable count of Agrobacterium sp. 1502IPR-08 in the bacterial agent is not less than 1×10⁻⁶. 9 CFU / mL.

[0010] This invention provides the application of the aforementioned microbial agent in the preparation of plant growth promoters, plant photosynthesis improvers, or plant nitrogen nutrition improvers.

[0011] This invention provides a bio-fertilizer, which includes the Agrobacterium sp. 1502 IPR-08, the fermentation product, or the microbial agent.

[0012] The present invention also provides the application of the Agrobacterium sp. 1502 IPR-08, the fermentation product, the inoculant, or the bio-fertilizer, wherein the application includes any of the following methods:

[0013] (1) Promote the conversion of nitrogen in the air into nitrogen nutrients that can be effectively absorbed by plants;

[0014] (2) Promotes plant assimilation of nitrogen in the soil;

[0015] (3) Enhance plant photosynthesis;

[0016] (4) Promote plant growth and development.

[0017] Preferably, the plants described in methods (1)-(4) include peanuts and / or corn.

[0018] Beneficial effects:

[0019] This invention isolates an Agrobacterium sp. 1502IPR-08 strain from plant rhizosphere soil, with the preservation number CGMCC No. 25883. This Agrobacterium 1502IPR-08 is a Gram-negative bacterium with highly efficient nitrogen-fixing capabilities, converting atmospheric nitrogen into nitrogen usable by plants and increasing soil nutrients. Experiments have shown that this Agrobacterium can increase the nitrogen content of rhizosphere soil, enhance the activity of glutaminase in plant roots, promote plant growth and development, and improve plant nitrogen nutrition. Furthermore, it can successfully colonize plant rhizosphere soil, and its application is pollution-free, residue-free, and environmentally friendly, demonstrating significant application potential in the field of plant growth promotion.

[0020] Biological Preservation Instructions:

[0021] Agrobacterium sp. 1502IPR-08 was deposited on October 9, 2022, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as Agrobacterium sp. 1502IPR-08, with accession number CGMCC No. 25883. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0023] Figure 1 This is a photograph showing the effect of Agrobacterium sp. 1502IPR-08 on peanuts in Example 2. The left side is the control group, and the right side is the treatment with Agrobacterium sp. 1502IPR-08.

[0024] Figure 2 This is a comparison chart showing how Agrobacterium sp. 1502IPR-08 improved the SPAD value of peanut leaves in Example 2.

[0025] Figure 3 This is a comparison chart showing how Agrobacterium sp. 1502IPR-08 improved peanut plant height in Example 2.

[0026] Figure 4 This is a comparison diagram showing how Agrobacterium sp. 1502IPR-08 enhanced the activity of glutamine synthase in peanut roots in Example 2.

[0027] Figure 5 This is a comparison chart showing how Agrobacterium sp. 1502IPR-08 increased the nitrogen content in peanut rhizosphere soil in Example 2. Detailed Implementation

[0028] This invention provides an Agrobacterium sp. 1502IPR-08.

[0029] The *Agrobacterium* sp. 1502IPR-08 provided in this invention was isolated from the rhizosphere soil of peanuts in a peanut-maize intercropping system, and named 1502IPR-08, hereinafter referred to as 1502IPR-08. This strain was deposited on October 9, 2022, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as *Agrobacterium* sp. 1502IPR-08, with accession number CGMCC No. 25883.

[0030] The colony and cell characteristics of strain 1502IPR-08 are as follows: colonies are round with a central protrusion and pale yellow in color. Microscopic observation shows that the cell morphology is rod-shaped, single-celled, Gram-negative, and without spores.

[0031] The experiments of this invention have found that the *Agrobacterium* sp. 1502 IPR-08 has the effects of efficient nitrogen fixation, improving plant nitrogen nutrition, increasing the nitrogen content of rhizosphere soil, and promoting plant growth. This is reflected in the fact that the growth-promoting bacterial preparation can increase the activity of glutamine synthase in plant roots and increase the nitrogen content of rhizosphere soil.

[0032] The fermentation product obtained by fermenting the above-mentioned Agrobacterium sp. 1502IPR-08 is also within the scope of protection of this invention.

[0033] In a preferred embodiment of the present invention, the fermentation medium used is LB medium: 5g yeast extract; 10g peptone; 10g sodium oxide; distilled water added to a final volume of 1000mL; pH = 7.0; sterilized at 121℃ for 20min; fermentation conditions are: temperature 30℃, rotation speed 150-200rpm. The fermentation product has the effects of efficient nitrogen fixation, improving plant nitrogen nutrition, increasing the nitrogen content of rhizosphere soil, and promoting plant growth.

[0034] This invention provides a microbial agent containing the aforementioned Agrobacterium sp. 1502IPR-08 or its fermentation product. Preferably, the microbial agent is a plant growth promoter, a plant photosynthetic improver, or a plant nitrogen nutrient improver.

[0035] Preferably, the microbial agent contains an effective viable count of Agrobacterium sp. 1502IPR-08 of not less than 1×10⁻⁶. 9 CFU / mL.

[0036] The present invention provides a bio-fertilizer containing the aforementioned Agrobacterium sp. 1502IPR-08 or its fermentation products.

[0037] This invention provides the application of Agrobacterium sp. 1502IPR-08 or its fermentation products, or inoculants or biofertilizers containing it, in promoting the absorption of nitrogen from the soil by plants. The Agrobacterium sp. 1502IPR-08 or its fermentation products, or inoculants or biofertilizers containing it, can promote the conversion of atmospheric nitrogen into nitrogen nutrients that can be effectively absorbed by plants.

[0038] This invention provides the application of Agrobacterium sp. 1502IPR-08 or its fermentation products, or inoculants or biofertilizers containing it, in improving the nitrogen fixation capacity of plant roots. The Agrobacterium sp. 1502IPR-08 or its fermentation products, or inoculants or biofertilizers containing it, can promote the assimilation of nitrogen from the soil by plants.

[0039] This invention provides the application of Agrobacterium sp. 1502IPR-08 or its fermentation products, or inoculants or biofertilizers containing it, in promoting plant growth. The Agrobacterium sp. 1502IPR-08 or its fermentation products, or inoculants or biofertilizers containing it, can enhance plant photosynthesis and promote plant growth and development.

[0040] In the above applications, the plant can be peanut and / or corn, preferably peanut. Those skilled in the art will understand that applying the 1502IPR-08 of the present invention to nitrogen absorption and growth promotion in other crops will also have similar growth-promoting effects.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are all conventional methods; the materials and reagents used are all commercially available.

[0042] Unless otherwise specified, the culture medium used in the embodiments of the present invention has the following formula: LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, diluted to 1L with distilled water, and the pH is adjusted to 7.0.

[0043] Example 1: Identification of the growth-promoting function of Agrobacterium sp. 1502 IPR-08

[0044] Identification of nitrogen fixation capacity of Agrobacterium sp. 1502IPR-08:

[0045] The test strain in the logarithmic growth phase was inoculated into LB liquid medium and cultured overnight at 30°C with shaking. After measuring the OD600 value of the strain, the cells were collected by centrifugation, washed three times with physiological saline, and then resuspended in nitrogen-limiting medium to adjust the OD600 to 0.2. 1 mL of bacterial suspension was added to each anaerobic tube, the tubes were sealed, air was removed, and 2.5 mL of freshly prepared acetylene was injected. The tubes were then cultured at 30°C with shaking for 72 h. 100 μL of gas was injected into each anaerobic tube using a microsyringe and the ethylene content was determined by gas chromatograph.

[0046] The formula for calculating enzyme activity is as follows:

[0047] Nitrogenase activity = Ethylene peak area on recorder × (gas volume in test tube / injection volume) / (peak area of ​​1 nmol standard ethylene × reaction time)

[0048] The results of nitrogenase activity assay for the strain are shown in Table 1. The results indicate that *Agrobacterium sp.* 1502IPR-08 has a strong nitrogen-fixing capacity, reaching 1.24 μmol / (h·ml).

[0049] Identification of the phosphorus solubilizing ability of Agrobacterium sp. 1502IPR-08:

[0050] Single colonies were inoculated onto Monkina inorganic phosphorus medium and cultured on a shaker at 28°C and 180 rpm for two days. The bacterial suspension was centrifuged at 10,000 rpm for 10 min, and 5 mL of the supernatant was collected, filtered through a 0.22 μm filter membrane, and diluted. 5 mL of the diluted solution was added to a 50 mL volumetric flask, followed by 5 mL of molybdenum antimony colorimetric solution, and deionized water was added to the mark. After standing for 30 min, the absorbance (OD880) at 880 nm was measured. A phosphorus concentration standard curve was plotted, and the phosphorus content dissolved from the bacterial suspension was calculated based on the phosphorus standard curve and the OD880 value of the bacterial suspension.

[0051] The results of the phosphorus solubilization capacity assay are shown in Table 1. The results indicate that *Agrobacterium sp.* 1502IPR-08 exhibits a strong phosphorus solubilization capacity, reaching 8.65 μg / mL.

[0052] Identification of the ability of Agrobacterium sp. 1502IPR-08 to produce glutathione:

[0053] Tryptophan was sterilized separately and added to LB liquid medium to a final concentration of 100 mg / L. Single colonies were inoculated and cultured on a shaker for 1 day at 28°C and 180 rpm. 1 mL of bacterial suspension was centrifuged at 10,000 rpm for 10 min, and 100 μL of the supernatant was dropped onto a white spot plate. Blank medium and 50 mg / L IAA solution were used as negative and positive controls, respectively. An equal volume of Salkowski chromogenic solution was added to each control, and the plate was incubated at room temperature in the dark for 30 min. 1 mL of the supernatant was mixed with an equal volume of Salkowski chromogenic solution, and the mixture was placed in a 40°C water bath in the dark for 30 min. The absorbance at 530 nm was measured colorimetrically, and the OD600 value of the bacterial suspension was also determined. Using the IAA concentration standard curve, the amount of IAA produced per unit volume of bacterial suspension when OD600 = 1 was calculated.

[0054] The results of the auxin production capacity assay are shown in Table 1. The results indicate that *Agrobacterium sp.* 1502IPR-08 has a strong auxin secretion capacity, reaching 3.08 μg / mL.

[0055]

[0056] Example 2: Agrobacterium sp. 1502 IPR-08 activates soil nitrogen, promoting plant nitrogen assimilation and plant growth.

[0057] The strain 1502IPR-08 of this invention was cultured in LB liquid medium at 30°C for 10... 9 The concentration of CFU / ml was centrifuged at 5000 rpm for 10 min in a high-speed centrifuge. The supernatant was discarded, and an equal volume of sterile water was added to prepare a bacterial suspension for later use.

[0058] On May 10, 2022, peanuts of the variety Luhua 14 were transplanted in the greenhouse of the College of Resources and Environment, China Agricultural University, Beijing. Starting from the first week after thinning, 50 ml of bacterial solution was added weekly per plant. The bacterial solution concentration during the treatment was 1×10⁻⁶. 9 CFU / ml, with an equal volume and concentration of sterile saline solution added to the control group. Four pots were used in each treatment group, with six biological replicates per pot for peanuts and three biological replicates per pot for maize. Peanuts were harvested three months after transplanting. One week before harvesting, the SPAD value of peanut leaves was measured using a SPAD meter to characterize their photosynthetic capacity. After harvesting, the plant samples were dried.

[0059] See results Figures 1-5 . Figures 1-5The results showed that the application of Agrobacterium sp. 1502IPR-08 significantly increased the nitrogen content in the rhizosphere soil of peanuts, promoted the assimilation of nitrogen by peanuts, improved the nitrogen deficiency chlorosis phenomenon in peanuts, and promoted the growth and development of peanuts. The main findings were as follows: The nitrogen content in the peanut rhizosphere soil of the control group was 0.047 g / kg, while that of the group treated with *Arbus suberectus* was 0.053 g / kg, representing a 12.77% increase compared to the control group. The glutamine synthase activity in peanut roots of the control group was 6.00 U / g, while that of the group treated with *Arbus suberectus* was 10.48 U / g, representing a 74.7% increase compared to the control group. The SPAD value of peanut leaves in the control group was 40.05, while that of the group treated with *Arbus suberectus* was 45.57, representing a 13.8% increase compared to the control group. The plant height in the control group was 30.06 cm, while that in the group treated with *Arbus suberectus* was 33.58 cm, representing an 11.7% increase compared to the control group, all of which were statistically significant. The results above lead to the conclusion that applying Agrobacterium sp. 1502IPR-08 to the plant rhizosphere can effectively improve the absorption and utilization of nitrogen nutrients, enhance photosynthetic capacity, and promote plant growth.

[0060] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention. However, they should not be construed as limiting the scope of patent protection. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Agrobacterium ( Agrobacterium sp. ) 1502IPR-08, characterized in that, The soil bacteria ( Agrobacterium sp. The accession number for 1502IPR-08 is CGMCC No.25883.

2. The *Agrobacterium* as described in claim 1 ( Agrobacterium sp. Fermentation broth of 1502IPR-08 or centrifuged resuspension of fermentation broth.

3. A microbial agent, characterized in that, The microbial agent includes the *Agrobacterium* as described in claim 1. Agrobacterium sp. )1502IPR-08 or the fermentation broth or centrifuged resuspension of the fermentation broth as described in claim 2.

4. The microbial agent according to claim 3, characterized in that, The bacterial agent contains Agrobacterium ( Agrobacterium sp. The effective viable count of 1502IPR-08 is not less than 1×10⁻⁶. 9 CFU / mL.

5. The use of the microbial agent according to claim 3 or 4 in the preparation of plant growth promoters, plant photosynthesis improvers, or plant nitrogen nutrition improvers; The plant is peanut and / or corn.

6. A bio-fertilizer, characterized in that, The biofertilizer includes the *Arthrobacter* as described in claim 1. Agrobacterium sp. ) 1502IPR-08 or the fermentation broth or centrifuged resuspension of the fermentation broth as described in claim 2 or the bacterial agent as described in claim 3 or 4.

7. The *Agrobacterium* as described in claim 1 ( Agrobacterium sp. The application of 1502IPR-08 or the fermentation broth or centrifuged resuspension of the fermentation broth according to claim 2, or the microbial agent according to claim 3 or 4, or the bio-fertilizer according to claim 6, is characterized in that... The application includes any of the following methods: (1) Promote the conversion of nitrogen in the air into nitrogen nutrients that can be effectively absorbed by plants; (2) Promotes plant assimilation of nitrogen in the soil; (3) Enhance plant photosynthesis; (4) Promotes plant growth and development; The plants mentioned in (1)-(4) are peanuts and / or corn.

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